1. Technical Field
The presence invention relates to a method and reagents for a fluorescence polarization immunoassay procedure for determining the presence or amount of benzoyl ecgonine and substituted benzoyl ecgonine compounds in fluids, especially biological fluids such as urine, serum or plasma, and to a method of making the reagents. More specifically, the invention relates to (1) reagents (tracers and antibodies) for determining the presence or amount of benzoyl ecgonine and substituted benzoyl ecgonine compounds in a sample; (2) immunogen compounds used to raise antibodies; (3) synthetic methods (for making tracers and immunogen compounds); and (4) analytical methods for conducting the assay.
2. Background Art
Cocaine is a very potent central nervous system stimulant. It is a naturally occurring alkaloid found in the leaves of the shrub species Erythroxylon and Erythroxylaceae. It historically has been used as a local anesthetic, but due to its stimulant properties, abuse has lead to increasing efforts to police its use and prevent its unauthorized distribution. These efforts are supported by detection methods that are rapid, reliable and selective for cocaine and/or cocaine metabolites. Cocaine is not highly toxic but is addictive. Cocaine is rapidly metabolized. In blood and urine the two major metabolites are benzoyl acgonine and ecgonine methyl ester, neither of which is pharmacologically active. Detection of either or both of these metabolites indicates cocaine use.
The most frequent biological fluid tested is urine. Urine samples are more accessible than blood samples, and other biological fluids have not been extensively investigated for use in assays.
In the past, urine samples have been tested for the presence of cocaine and cocaine metabolite by thin layer chromatography, enzyme immunoassay, gas chromatography or high performance liquid chromatography (HPLC) assays. These methods are not without drawbacks; for example, the assay time can typically be lengthy.
In assays for other substances, competitive binding immunoassays have provided a more satisfactory alternative. Typically, competitive binding immunoassays are used for measuring ligands in a test sample. (For the purposes of this disclosure, a "ligand" is a substance of biological interest to be quantitatively determined by a competitive binding immunoassay technique.) The ligands compete with a labeled reagent, or "ligand analog" or "tracer" for a limited number of receptor binding sites on antibodies specific to the ligand and ligand analog. The concentration of a ligand in the sample determines the amount of ligand analog which binds to the antibody; the amount of ligand analog that will bind is inversely proportional to the concentration of ligand in the sample, because the ligand and the ligand analog each bind to the antibody in proportion to their respective concentrations.
Fluorescence polarization provides a quantitative means for measuring the amount of tracer-antibody conjugate produced in a competitive binding immunoassay. Fluorescence polarization techniques are based on the principle that a fluorescent labeled compound, when excited by plane polarized light, will emit fluorescence having a degree of polarization inversely related to its rate of rotation. Accordingly, when a tracer-antibody conjugate having fluorescent label is excited with plane polarized light, the emitted light remains highly polarized because the fluorophore is constrained from rotating between the time that light is absorbed and emitted. In contrast, when an unbound tracer is excited by plane polarized light, its rotation is much faster than the corresponding tracer-antibody conjugate and the molecules are more randomly oriented. As a result, the light emitted from the unbound tracer molecules is depolarized.
A problem that heretofore has prevented the accurate determination of cocaine and other "drugs of abuse" in urine by fluorescence polarization techniques is that of riboflavin interference. Riboflavin, or vitamin B.sub.2, is a common constituent of many foods and of commercially available vitamin supplements. Riboflavin is excreted primarily in the urine and has a fluorescence spectrum quite similar to that of fluorescein. As as result, the presence of riboflavin in even moderate amounts in urine samples creates an interference which can produce erroneous data. While ordinary consumption of riboflavin is unlikely to produce more than trace amounts of riboflavin in the urine, test results can readily be distorted by the consumption of excessive quantities of vitamin supplements by persons wishing to prevent detection of cocaine use.
The present invention offers an advance in the art in that highly sensitive tracers, a method for making the tracers, and an assay using the tracers are provided specifically for the determination of benzoyl ecgonine and substituted benzoyl ecgonine compounds without riboflavin interference.